The p53 protein prevents tumor formation by inhibiting cell proliferation following cellular stress. It is not surprising therefore that nearly every human tumor possesses a disruption in the p53 pathway. While clinical approaches to reactivate p53 activity may improve current cancer treatments, there still remain important questions regarding how cellular factors affect the p53 network. Our group has been studying the role of MdmX in regulating p53 function. Like its partner Mdm2, MdmX represents an important p53 inhibitor during development. However how MdmX deregulation affects the p53 pathway and cellular transformation in adult cells represent aspects of MdmX and p53 requiring further study. Aim 1 will test the hypothesis that MdmX overexpression leads to cell transformation and increased cellular proliferation. Using primary and immortalized human and mouse cells, we will assess the effects of MdmX overexpression and knock-down (RNAi) on cellular transformation, proliferation and senescence. DNA microarray analysis will be employed to assess the cellular signals altered by MdmX deregulation. The second aim will examine current models of how MdmX:Mdm2 and MdmX:ARF interactions affect the p53 pathway to address the hypothesis that MdmX can alter p53 activity through direct interactions with the p53 regulators Mdm2 or ARF. This aim will assess the interdependency of MdmX and Mdm2 in regulating p53 function, determine how MdmX localization affects its regulation of p53 and will continue present studies of how MdmX proteins that specifically target Mdm2 enhance p53 activity. Lastly, using RNAi approaches we will examine how MdmX:ARF interactions impact ARF activities relating to Mdm2 and p53. The third and final aim will explore p53-independent effects of MdmX deregulation. Based in part on our recent report that MdmX inhibits E2F1 transactivation in a p53- independent manner, we will test whether MdmX deregulation affects the activity of other E2F members, E2F DNA binding in vivo and E2F1-dependent apoptosis. Lastly, the impact of MdmX deregulation in human cells lacking p53 will also be investigated. Overall this proposal will employ cellular and molecular appproaches to address specific hypotheses and questions resulting in an improved understanding of MdmX deregulation in tumorigenesis. This knowledge may ultimately lead to improved therapeutic approaches to reactivate the p53 pathway in human cancers.